1. Stroma-Free Hemoglobin
Intravenously injected (infused) crude hemolysates and extensive hemolytic processes produced in vivo by immunological reactions involving intravascular lysis of red blood cells, are known to produce a clinical syndrome characterized by disseminate intravascular coagulation. This syndrome is often fatal and is produced by the residual red blood cell walls (stroma) and their fragments, so infused into circulating blood. Stroma-free hemolysates do not show this toxicity (See Rabiner, S. F. et al, J. Exp. Med., 126:1127 (1967)). As a result, it has been desired to use stroma-free hemoglobin as an oxygen carrier in cell-free transfusional fluids.
However, the use of stroma-free hemoglobin has the following two disadvantages. In vivo, the retention time of the stroma-free human hemoglobin is very short, i.e., it has a half-life on the order of 1-4 hours (See Rabiner, S. F., supra, and De Venuto, F. et al, Transfusion, 17:555 (1977)). "Half-life" is defined as the time necessary to eliminate 50% of the infused hemoglobin from circulating blood. Further, outside of the red blood cells, hemoglobin has a high affinity for oxygen which, in vivo, would prevent the release, i.e., the transport, of oxygen from hemoglobin to the tissues. These disadvantages are directly the result of the molecular structure of hemoglobin.
Hemoglobin is a tetrameric molecule having a molecular weight of 64,500 Daltons. The tetrameric molecule is formed of two pairs of alpha and beta subunits. The subunits are held together as a result of ionic and Van der Waals forces, and not as a result of covalent bonds. When hemoglobin is oxygenated, i.e., combined with oxygen, it readily forms alpha-beta dimers having a molecular weight of 32,250 Daltons. These dimers are not retained in vivo by the kidneys and are eliminated through the urine.
The tetrameric structure of hemoglobin also provides a binding site for 2,3-diphosphoglycerate. Inside red blood cells, 2,3-diphosphoglycerate combines with hemoglobin in order to decrease its oxygen affinity to a level compatible with oxygen transport. The binding of 2,3-diphosphoglycerate and hemoglobin is purely electrostatic and no stable covalent bonds are formed. Thus, when red blood cells are ruptured and 2,3-diphosphoglycerate is not retained inside the cells by the cell wall, it is released from hemoglobin. As a result, hemoglobin acquires a higher oxygen affinity. This prevents the transport of oxygen from hemoglobin to the tissues. The level of this higher affinity is sufficient such that the oxygen affinity can be considered "non-physiological".
Because of the many appealing qualities of hemoglobin i.e., its ability to reversibly bind oxygen, the low viscosity of a hemoglobin solution and its easy preparation and storage for long periods of time, various attempts have been made in order to overcome the above-described disadvantageous characteristics of stroma-free hemoglobin. These various attempts are discussed in more detail below.
2. Chemical Treatments for Preventing the Formation of Dimers
The formation of alpha-beta dimers, which are not retained in vivo, can be prevented by coupling the tetrameric molecules of hemoglobin with large molecular weight matrices, ranging from 20,000 to 275,000 Daltons, for example, matrices such as dextran (See Tam, S. C. et al, Can. J. Biochem., 56:981 (1978) and Bonneaux, F. et al, Experientia, 37:884 (1981)) and hydroxyethyl starch (See Baldwin, J. E. et al, Tetrahedron, 37:1723 (1981)). This coupling prevents the elimination of hemoglobin in vivo from the kidneys by way of the urine. Other types of polymeric coupling employing collagen, collagen degradation products, and gelatin as a supporting matrix have also been employed (See U.S. Pat. No. 2,591,133, U.S. Pat. No. 3,057,782, and Bowes, F. et al, Biochem. Biophys. Acta, 168:341 (1968)). However, the oxygen affinity of the resulting coupled hemoglobin is even higher than that of stroma-free hemoglobin and thus hemoglobin coupled in this manner cannot be advantageously employed as an oxygen transport medium.
Other known treatments for preventing the formation of alpha-beta dimers are based on reactions which polymerize the tetrameric molecules of hemoglobin to form so-called "polyhemoglobins". Polyhemoglobins can be obtained using bifunctional reagents such as gluteraldehyde (See Hopwood, C. et al, Histochem. J., 2:137 (1970) or diimidate esters (See Mock, W. et al, Fed. Proc., 34:1458 (1975)) and U.S. Pat. No. 3,925,344). These bifunctional reagents form covalent bonds between the amino groups present on the surface of different hemoglobin molecules producing intermolecular cross-links. There are 40 or more of such amino groups belonging to lysyl residues on the surface of mammalian hemoglobins (44 in human hemoglobin) so that a large number of possible combinations of hemoglobin molecules occur. As a result, the polyhemoglobin reaction products are a heterogeneous mixture of various molecular species which differ in size and shape. The molecular weights thereof range from 64,500 to 600,000 Daltons. The separation of individual molecular species from the heterogeneous mixture is virtually impossible. In addition, although longer retention times in vivo are obtained using polyhemoglobins, the oxygen affinity thereof is higher than that of stroma-free hemoglobin.
Besides the various treatments discussed above which result in formation of heterogeneous mixtures of polyhemoglobin, reagents have been developed which are capable of producing an internal cross-link of the hemoglobin subunits with little or no formation of polyhemoglobins. More specifically, the formation of cross-links between the beta subunits of hemoglobin using 2-N-2-formyl-pyridoxal-5'-phosphate and borohydride has been carried out (See Bensch, R. et al, Biochem. Biophys. Res. Comm., 62:1123 (1975)). The oxygen affinity of the thus treated hemoglobin is decreased to levels similar to that of normal blood. However, the reagent employed therein is very difficult and costly to synthesize, and thus the method is disadvantageous.
Other reagents have been employed in order to effect internal cross-linking of the hemoglobin subunits. These reagents are commonly known as "diaspirins". Diaspirins are diesters of bis-3,5-dibromosalicylate containing succinyl, fumaryl or other dicarboxylic acid residues. These reagents produce covalent cross-links between two beta or two alpha subunits of an individual hemoglobin molecule. While better results are obtained using liganded (oxy- or carboxy-) hemoglobin, such a treatment does not sufficiently affect the oxygen affinity characteristics of stroma-free hemoglobin and thus cannot be advantageously employed. (See Walder, J. A. et al, J. Mol. Biol., 141:195 (1980), U.S. Pat. No. 4,061,736, U.S. Pat. No. 4,001,200, U.S. Pat. No. 4,001,401, and U.S. Pat. No. 4,053,590)).
In U.S. Pat. No. 4,473,496, linear alpha-omega or heterocyclic polyaldehydes containing negatively charged groups are described as suitable for both decreasing the oxygen affinity of hemoglobin and for producing inter- and intramolecular cross-linking of hemoglobin. These reagents include carbohydrate-containing molecules such as mono- and polyphosphorylated nucleotides partially oxidized with periodate, so as to obtain aldehydic groups. The coupling reaction is based on the formation of Shiff bases of the aldehydic groups with the amino groups of the hemoglobin molecule. The Shiff bases are then transformed into covalent bonds by reduction with sodium or potassium borohydride, or another strong reducing agent.
The above-described treatments, i.e., those based on the use of aldehydic reagents, have a further disadvantage in that they must be performed in the absence of air and oxygen, i.e., they must be performed on deoxyhemoglobin. That is, in the presence of air and oxygen, the reaction does not occur or does not produce the desired effects on the characteristics of hemoglobin. Also, oxygen-absorbing chemicals, e.g., sodium dithionite, cannot be used because they interfere with the reagents used for the treatment. Thus, these treatments must be disadvantageously performed in a closed environment and oxygen is removed either by flushing and/or bubbling the solution with nitrogen or some other inert gas, or using mechanical evacuation, or combining the two procedures. These treatments always produce disadvantageous denaturation of 2 to 5% of the hemoglobin present, i.e., denatured hemoglobin irreversibly loses its ability to combine with and transport oxygen.
3. Chemical Treatments for Decreasing the Oxygen Affinity of Stroma-Free Hemoglobin
The most widely used chemical modification of stroma-free hemoglobin so as to decrease the oxygen affinity thereof employs the use of pyridoxal-5'-phosphate and sodium or potassium borohydride (see Bensch, R. et al, Biochem., 11:3576 (1972)). The resulting product is commonly referred to as "PLP-hemoglobin" and has satisfactory oxygen affinity, i.e., oxygen affinity very near that of the red cells present in normal blood. However, in order to be effective, this treatment must be performed on deoxy-hemoglobin i.e., hemoglobin devoid of oxygen. Thus, this procedure must disadvantageously be carried out in a closed environment in the absence of air and oxygen as described above for reactions involving aldehydic groups.
Other known chemical modifications of hemoglobin have been carried out using phosphoric acid derivatives of carbohydrates (e.g. glucose-6-phosphate) (See McDonald, M. J. et al, J. Biol. Chem., 254:702 (1979)); carbamylation (See Manning, J. S. Meth. Enz., 76:159 (1981) and carboxymethylation (See DiDonato, A. et al, J. Biol. Chem., 258:11890 (1983)). In each of these treatments, the amino-terminal end of the beta subunit of hemoglobin is permanently substituted with the above-described reagents.
In addition, none of these chemical treatments discussed in this part stabilize the tetrameric structure of hemoglobin so as to prevent the formation of alpha-beta dimers. Thus, the resulting hemoglobins do not have prolonged retention times in vivo.
4. Combined Chemical Treatments for Preventing the Formation of Alpha-Beta Dimers and Decreasing the Oxygen Affinity of Stroma-Free Hemoglobin
As discussed above, the production of physiologically competent stroma-free hemoglobin-based oxygen carriers necessitates two separate treatments. That is, one treatment is necessary for preventing the formation of alpha-beta dimers in vivo and a second treatment is required for decreasing its oxygen affinity. The most widely employed combination of treatments is that of reacting gluteraldehyde with PLP-hemoglobin to form pyridoxylated polyhemoglobins (See Seghal, L. K. et al, J. Surg. Res., 30:14 (1981)). Intramolecular cross-linking of PLP-hemoglobin has also been obtained using diaspirins (See, Tye, R. et al, Prog. Clin. Biol. Res., 22:41 (1983)).
Again, the above-described chemical treatments must be disadvantageously performed on deoxyhemoglobin in the absence of oxygen or air in closed containers under, for example, nitrogen or some other inert gas with or without mechanical evacuation without the aid of oxygen-absorbing chemicals. Otherwise, hemoglobin with very high oxygen affinity is obtained, i.e., higher than that of stroma-free hemoglobin.
It should be noted that only stroma-free hemolysates or washed red blood cells are utilized in the above-cited articles. That is, purification procedures for isolating the hemoglobin component of the stroma-free hemolysates are not described therein. Thus, what is defined as stroma-free hemoglobins therein is in actuality stroma-free hemolysates.
More specifically, about 95% of the hemolysate components is hemoglobin. The remainder consists of proteins and polypeptides whose pharmacological and immunological toxicity is not known. When used for infusion in animals, several grams of hemolysate-containing hemoglobin are injected. Thus, undesirably, hundreds of milligrams of substances of unknown biological activity are also infused into animals when employing a hemolysate.
It should also be noted that in the above-cited references, purification procedures for isolating the desired hemoglobin products from the reaction mixture are not described therein. It is impossible to avoid the presence of overreacted and underreacted hemoglobins in the reaction mixtures. These products do not have the desired functional and molecular characteristics.
For the above reasons, it is advantageous to perform chemical treatments on purified hemoglobins, and then to purify the product of the reaction.